A Rapid, Reliable RP-UPLC Method for Large-Scale Analysis of Wheat HMW-GS Alleles
| Autor: | Sun-Hyung Lim, Yu-Jeong Yang, Yong Q. Gu, Su-Bin Lee, Jong-Yeol Lee |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Germplasm
large wheat germplasm Glutens Flour Wheat flour Gene Expression Pharmaceutical Science Biology High-performance liquid chromatography Article Analytical Chemistry chemistry.chemical_compound QD241-441 Glutenin Gene Frequency Gene Expression Regulation Plant Drug Discovery Storage protein Cultivar Physical and Theoretical Chemistry Allele Alleles Triticum chemistry.chemical_classification Chromatography Reverse-Phase Chromatography Organic Chemistry food and beverages HMW-GS RP-UPLC Molecular Weight allelic analysis Plant Breeding Protein Subunits chemistry Chemistry (miscellaneous) biology.protein Molecular Medicine Electrophoresis Polyacrylamide Gel Transcriptome DNA |
| Zdroj: | Molecules Volume 26 Issue 20 Molecules, Vol 26, Iss 6174, p 6174 (2021) |
| ISSN: | 1420-3049 |
| DOI: | 10.3390/molecules26206174 |
| Popis: | High-molecular-weight glutenin subunits (HMW-GS) account for only 10% of total wheat storage proteins, but play an important role in the processing quality of wheat flour. Therefore, identifying HMW-GS alleles associated with good end-use quality provides important information for wheat breeders. To rapidly, accurately and reproducibly identify HMW-GS, we established an optimized reversed-phase ultra-performance liquid chromatography (RP-UPLC) method. Separation parameters were optimized using an ACQUITY UPLC Protein BEH C4 column and stepwise ACN gradient, and the separation patterns and retention times (RTs) of 22 subunits were comparatively analyzed in 16 standard wheat cultivars. All HMW-GS proteins were well separated within about 5.5 min, and all analyses were complete within 12 min. We distinguished the 16 subunits based on RT, although three subunits in 1Bx (1Bx7/1Bx7OE and 1Bx17) and three subunits in 1By (1By8*, 1By9 and 1By15) had overlapping RTs these were differentiated by SDS-PAGE. To distinguish 1Bx7 and 1Bx7OE, which differ in protein abundance, RP-UPLC was combined with PCR analysis of DNA junction markers. The optimized method was successfully applied to determine HMW-GS alleles in a large collection of bread wheat germplasm (1787 lines). This protocol is an appropriate option for selecting lines harboring favorable HMW-GS alleles in wheat breeding. |
| Databáze: | OpenAIRE |
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